The present invention relates to the field of environmental scanning electron microscopes, and methods for using the same.
Conventional Scanning Electron Microscopes (CSEMs) require most samples to be dried of all water, and then coated with metal or carbon. This treatment generally precludes the study of dynamic events, such as the effects of dissolution.
In contrast, Environmental Scanning Electron Microscopes (ESE microscopes) and similar variable pressure microscopes, allow samples with a high moisture content to be imaged. Within an ESE Microscope, the samples are imaged by introducing water vapor into the chamber, and ionizing the vapor cloud directly over the sample. By controlling both the chamber pressure and the sample temperature, the sample can be maintained in a water saturated state.
In order to evaluate the effect of a substance on a sample, it is desirable to view a single sample at various time intervals after being exposed to the substance in a dissolution bath. For example, the dissolution characteristics of controlled released pharmaceuticals are often critical to the pharmaceutical""s usefulness. Moreover, it is often important to monitor the dissolution of controlled release pharmaceuticals for extended time periods (e.g. 8, 12, or 24 hours or more).
Since the controlled release pharmaceuticals are moist during dissolution, it is advantageous to view these pharmaceuticals using an ESE microscope or other variable pressure microscope. This approach, however, has a number of drawbacks. First, the controlled release pharmaceutical sample is subject to damage when it is transferred from the dissolution bath to the ESE microscope. Second, once a sample of the pharmaceutical is removed from the dissolution bath for viewing with the ESE microscope, it can not be returned to the dissolution bath.
To alleviate these problems, conventional ESE microscope""s offer a xe2x80x9cpeltier stagexe2x80x9d which is mounted in the ESE microscope specimen chamber and which allows moisture to be condensed onto a sample by controlling the temperature of the peltier stage. In this manner, the peltier stage can be used to provide a xe2x80x9cdissolution bathxe2x80x9d of water for a sample. The peltier stages, however, are inadequate for evaluating the dissolution characteristics of pharmaceuticals for a number of reasons.
For example, current peltier stages are too small to hold a pharmaceutical tablet and, since they operate by condensing moisture onto the sample from the atmosphere within the ESE microscope, they cannot provide the desired degree of xe2x80x9cmixingxe2x80x9d for an effective dissolution experiment. In addition, since they operate on a condensation principle, it is not possible to use these stages to conduct dissolution experiments with other dissolution media, such as simulated gastric fluid or simulated intestine fluid.
Moreover, in order to conduct a dissolution experiment with a peltier stage, the ESE microscope must first cool the stage so that enough water condenses into the sample well of the peltier stage to immerse the sample in water. Then, in order to image the sample, the stage must be heated sufficiently to evaporate the water in the well so that the sample can be imaged. This process has a number of disadvantages. First, rather than allowing the sample to be maintained at a desired temperature (for example, 98.6xc2x0 F., 37xc2x0 C.) throughout the experiment, the sample must be repeatedly cooled to cause condensation, and then heated to cause evaporation. As a result, it is not possible to simulate the dissolution experiment of the human body. In addition, the condensation/evaporation technique becomes increasingly impractical as the size of the sample, and therefore the amount of water to be condensed and evaporated, is increased.
It is also known to deposit a sample into a sample cup located in the ESE microscope Specimen chamber, and to introduce liquid into a sample cup by using a syringe or similar device. Such a method, however, also fails to provide the desired degree of mixing, and, moreover, is inadequate for long term automated experiments because an operator must be present to refill the sample cup with liquid. Moreover, since this technique requires removal of the water by evaporation, it suffers from the same deficiencies as the peltier stage described above.
In accordance with the present invention, a system is provided for imaging, in an ESE microscope or other variable pressure microscope, a single sample at various time intervals during dissolution of the sample in a liquid. The system includes a sample chamber having a sample well. The sample well includes an first fluid port and a second fluid port for forming a dissolution bath in the sample well. In accordance with the system according to the present invention, the sample chamber is placed into the specimen chamber of the ESE microscope and a sample is deposited into the sample well of the sample chamber. Preferably, the sample well is large enough to fully immerse a typical pharmaceutical sample which is prepared as a solid oral dosage form (e.g. tablets from  less than 5 mg to 1000 mg). The sample is immersed in a liquid which flows through the sample well via the first and second fluid ports during a dissolution cycle. The liquid is then drained from the sample well via one of the first and second fluid ports during a draining cycle, and then, during an imaging cycle, the sample is imaged by the ESE microscope. The dissolution cycle, the draining cycle, and the imaging cycle all occur while the sample well is inside the specimen chamber of the ESE microscope. By immersing the sample in a flowing liquid, a mixing effect is achieved which promotes dissolution of the sample because it reduces or eliminates the boundary zones which would otherwise form around the sample and impede dissolution. Moreover, since the sample well is filled and drained while it remains in the specimen chamber, a single sample can be imaged at various stages of dissolution by draining the well, imaging the sample, and then refilling the well at predetermined time intervals. In addition, the sample chamber in accordance with the present invention is not limited to using water as the dissolution fluid. Other dissolution media, such as simulated gastric fluid or simulated intestine fluid, can also be used.
Preferably, the second fluid port of the sample well is elevated relative to the first fluid port. This construction provides a number of additional advantages including i) preventing overflow of the well; and ii) providing a xe2x80x9csippingxe2x80x9d effect which causes the level of water in the well to rise and fall, thereby enhancing the mixing effect. In accordance with this embodiment, the sample well is filled by coupling a source of dissolution fluid to the first fluid port during the dissolution cycle, and then coupling the first fluid port to a drain line during the draining cycle to drain the fluid from the sample well. A vacuum source (such as a pump) could also be coupled to the drain hose to more quickly and effectively drain the fluid from the sample well. This can be implemented in any known manner. For example, a three port valve could be used, with one port coupled to a water faucet, one port connected to a drain hose, and the other port connected to the first fluid port of the sample well. The valve could then be actuated in any known manner to couple the water faucet to the input port during the dissolution cycle, and to couple the drain hose to the first fluid port during the draining and imaging cycles. The valve could be actuated mechanically or electrically (or in any other known manner), and the actuation could be triggered manually by the operator, or automatically via, for example, a computer or other automatic control system.
In accordance with a further aspect of the invention, a passage at least partially surrounds the sample well, and the passage is coupled to a heating and/or cooling source to provide for temperature control of a sample placed in the sample well. Preferably, water is used as the heating and cooling medium. This construction provides excellent heat transfer characteristics and allows large samples to be quickly heated and cooled.
In accordance with another embodiment of the invention, the sample chamber includes a movable lid which covers the sample well during the dissolution cycle, and exposes the sample well during the imaging cycle in order to allow imaging of the sample. In general, an ESE microscope seeks to maintain the pressure in the specimen chamber at a specified level. If the sample well of the sample chamber is uncovered during the dissolution cycle, water will evaporate into the specimen chamber, and alter the pressure in the specimen chamber. Upon detecting the change in pressure, the ESE microscope will utilize its pumps to increase or decrease the pressure until specified pressure level is attained. This causes an undesirable strain on the pumps, which are not designed to compensate for the relatively large amount of water which evaporates during the dissolution cycle. Therefore, by providing a movable lid for the sample chamber, the strain on the ESE microscope""s pumps is reduced. Alternatively, the microscope""s vaccum pumps could be set to standby, eliminating the need to place a lid on the well.
In accordance with a still further embodiment of the invention, the system is configured to run long term automated dissolution experiments. In accordance with this embodiment, the system includes a controller, an ESE microscope, a sample chamber, and an image storage device. The image storage device and controller can be of any known construction. For example, the image storage device could be a VCR or a computer, and the controller could be a computer or even a simple programmable timer.
This construction allows an operator to perform in-chamber dissolution experiments with a variety of dissolution media, provides improved thermal control of larger samples, eliminates the mixing problems associated with prior art stage baths, allows for long running experiments (e.g., 8, 12, 24 hrs. or more) with increased automation, provides automatic image capture during long running experiments, and protects the ESE microscope from excessive amounts of moisture during non-imaging periods.